WO2005073154A1 - Procede d'extraction de totarol et/ou produit contenant du totarol - Google Patents

Procede d'extraction de totarol et/ou produit contenant du totarol Download PDF

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Publication number
WO2005073154A1
WO2005073154A1 PCT/NZ2005/000008 NZ2005000008W WO2005073154A1 WO 2005073154 A1 WO2005073154 A1 WO 2005073154A1 NZ 2005000008 W NZ2005000008 W NZ 2005000008W WO 2005073154 A1 WO2005073154 A1 WO 2005073154A1
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Prior art keywords
totarol
extract
oil
critical fluid
containing extract
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PCT/NZ2005/000008
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English (en)
Inventor
Douglas Anthony Mende
Owen John Catchpole
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Douglas Anthony Mende
Owen John Catchpole
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Application filed by Douglas Anthony Mende, Owen John Catchpole filed Critical Douglas Anthony Mende
Publication of WO2005073154A1 publication Critical patent/WO2005073154A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/12—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings
    • C07C39/17—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings containing other rings in addition to the six-membered aromatic rings, e.g. cyclohexylphenol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9755—Gymnosperms [Coniferophyta]
    • A61K8/9761—Cupressaceae [Cypress family], e.g. juniper or cypress
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9755—Gymnosperms [Coniferophyta]
    • A61K8/9767—Pinaceae [Pine family], e.g. pine or cedar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/524—Preservatives

Definitions

  • This invention relates to a method of producing a totarol-containing extract from suitable plant material and to uses of, and compositions comprising, the extract.
  • Totarol (totara-8,1 l,13-trien-13-ol; 4b-S-tr ⁇ «5 , -8,8-trimethyl-4b,5,6,7,8,8a,9,10-octahydro-l- isopropylphenanthren-2-ol), is an aromatic diterpenoid natural product with the molecular formula C 20 H 30 O.
  • Totarol is present in relatively high concentrations — approximately 5 % by mass on a dry basis — in the heartwood of Totara (Podocarpus Totara), which is indigenous to New Zealand; and in much lower concentrations — less than 1 % by mass — in other Podocarpus, Dacrycarpus, Cupressus and Juniperus species.
  • Totarol is remarkably resistant to oxidation and degradation and so the level in dead heartwood remains relatively constant despite the age of the timber.
  • Totarol can be produced synthetically, by a number of routes, but the economics and suitability of these methods have not been proven to be feasible for production on a large scale, and often both stereoisomers are produced. Therefore, production of totarol for pharmaceutical, nutraceutical, cosmetic, cosmeceutical and other applications is commercially possible only by extraction of the naturally occurring compound from suitable plant raw material.
  • Totarol is known to be soluble in the common organic solvents hexane, acetone, ethanol and methanol.
  • these solvents are not selective, and removal of the solvent residues from the extracted material is generally incomplete, which makes their use particularly unattractive when the resulting totarol extracts are intended for use in pharmaceutical, nutraceutical, cosmetic and cosmeceutical applications.
  • Totarol is a potent antimicrobial agent against Gram-positive bacteria (1).
  • JP 01-311019 describes the topical use of totarol as an anti-bacterial agent against Gram-positive bacteria.
  • totarol has not previously been reported to have any activity against Gram- negative bacteria.
  • Tea-tree oil is an essential oil steam-distilled from the plant Melaleuca alternifolia, which is indigenous to Australia. Tea-tree oil is a complex mixture of approximately 100 terpenes and hydrocarbons, and is one of the few known all natural extracts that has antimicrobial activity against Gram-negative bacteria.
  • the antimicrobial activity of plant oils and extracts may be measured as the minimum inhibitory concentration (MIC), which is the lowest concentration which results in the maintenance or reduction of inoculum viability. This is the most commonly reported method in the antimicrobial literature and may permit comparison between studies. Two methods of MIC determination are usually employed — the agar dilution method, in which various concentrations of the test substance are added to an agar medium prior to inoculation with the test organism; and the broth dilution method, in which the test organism is added to serial dilutions of the antimicrobial agent prepared in a nutrient medium.
  • MIC minimum inhibitory concentration
  • Table 1 sets out a comparison of the antimicrobial activity of tea-tree oil and totarol produced by solvent extraction against various bacteria.
  • this comparison is limited due to the insufficient amount of published MIC data for totarol against various organisms.
  • the few investigations that have been published may not be directly comparable because of the assay methods that were used i. e. broth dilution versus agar dilution.
  • the composition of the active components may also differ in proportion from sample to sample thus influencing the reproducibility of the results.
  • totarol produced by organic solvent extraction has greater antimicrobial activity than tea-tree oil against two micro-organisms — the Gram-positive bacteria, Enterococcus faecalis and Propionibacterium acnes.
  • totarol has significantly lower antimicrobial activity than tea-tree oil against the Gram- negative bacterium, Klebsiella pneumonia.
  • Table 1 MIC values for tea-tree oil and solvent-extracted totarol against various bacteria
  • the present invention provides a process for producing a totarol-containing extract from suitable plant material, which process comprises the steps of:
  • the present invention provides a process for producing a totarol- containing extract, which extract comprises totarol and an oil, from suitable plant material and suitable oil-containing material, which process comprises the steps of: (a) contacting the plant material and oil-containing material with a near-critical fluid to produce a near-critical fluid phase in which the totarol and oil is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.
  • the present invention provides a totarol-containing extract when produced by a process of the invention.
  • the present invention provides a totarol-containing extract, wherein the extract comprises one or more other compounds selected from the group consisting of: totarol hemiacetal; 7a-hydroxytotarol; 7-oxototarol; 5-hydroxytotarol; 6,7-dehydrototarol; and ferruginol.
  • the present invention provides an extract obtained from the heartwood of Podocarpus totara G. Benn (Totara) or Podocarpus Hallii (Halls Totara), wherein the extract has activity against Gram-negative bacteria.
  • the present invention provides a composition comprising an extract of the invention.
  • the present invention provides a product, comprising an extract of the invention as an active ingredient, antimicrobial additive, or preservative.
  • the present invention provides a therapeutic formulation having activity against Gram-positive and Gram-negative bacteria, wherein the formulation comprises an extract of the invention.
  • the present invention provides a use of an extract of the invention in the preparation of a therapeutic formulation for treating a Gram-negative bacterial infection in an individual in need thereof.
  • the present invention provides a method for at least inhibiting the growth of bacteria sensitive to an extract of the invention, the method comprising contacting the sensitive bacteria with an inhibitory effective amount of the extract, or a composition comprising the extract.
  • the present invention provides a method of prophylactic or therapeutic treatment of bacterial infection in an individual in need thereof, the method comprising administering to said individual an extract of the invention, or a composition comprising the extract, in an amount effective to at least inhibit growth of the bacteria.
  • This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • Figure 1 is a schematic diagram of an extraction apparatus which is suitable for use in performing a process of the invention.
  • an extract comprising totarol can be produced from suitable plant material by near-critical extraction, and that a carrier liquid infused with the totarol-containing extract may be produced directly by near-critical extraction. Furthermore, the applicants have determined that the totarol-containing extract has activity against both Gram-positive and Gram-negative bacteria.
  • the present invention provides a process for producing a totarol-containing extract from suitable plant material, which process comprises the steps of: (a) contacting the plant material with a near-critical fluid to produce a near-critical fluid phase in which the totarol is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.
  • contact generally means admixing the plant material with the near-critical fluid in a suitable extraction vessel using apparatus as are well known in the art.
  • the apparatus may be equipped with multiple extraction vessels, each of which may be removed from the flow of near-critical fluid without removing the others, thereby permitting emptying and refilling of the extraction vessels without depressurising the apparatus, enabling semi-continuous operation.
  • the term "separate" as used herein in relation to the near-critical fluid phase and the plant material generally means removing the stream comprising the near-critical fluid phase and the dissolved totarol from the apparatus, while excluding the plant material from the stream.
  • near-critical fluid means a fluid that is close to its critical point and thus includes both subcritical and supercritical fluids. Near-critical includes the reduced temperature range 0.75 ⁇ T r ⁇ 1.25 (where T r is the temperature divided by the critical temperature, T c of the fluid); and the pressure ranges P > P v (where P v is the vapour pressure) for T ⁇ T c and P > P c (where P c is the critical pressure) for T > T c .
  • Near-critical fluids are selective solvents that have found application in various extraction processes.
  • a near-critical fluid has a density comparable to that of a liquid while exhibiting the diffusion properties of a gas.
  • the density of the fluid is highly dependent on temperature and pressure.
  • the solvent power of the fluid is directly related to the density at fixed temperature, and temperature at fixed density.
  • the density of the fluid is high and the fluid can act as a solvent.
  • the pressure is lowered and/or the temperature increased, the density and, therefore, the solvent power of the fluid is reduced, and the material dissolved in the fluid can be separated from it.
  • Preferred near-critical fluids include, but are not limited to: CO 2 ; C 2 -C 4 hydrocarbons, including ethane, ethylene, propane, propylene, and butane; partially and fully fluorinated Ci-C 3 hydrocarbons, particularly R134a (1,1,1,2-tetrafluoroethane); iodotrifluoromethane; nitrous oxide; sulfur hexafluoride; dimethylether; and mixtures of any two or more of the above.
  • CO 2 is widely used as a supercritical fluid as its critical temperature and pressure (31.2°C, 73.2 bar) are attained relatively easily. Furthermore, CO is inert, non-toxic, cheap and readily available.
  • the near-critical fluid used is CO 2 .
  • liquid CO 2 is used (T ⁇ 31.2 °C, pressure greater than the vapour pressure of CO 2 at T).
  • the plant material is contacted with supercritical CO 2 at a desired pressure (> 75 bar) and desired temperature, T (> 31.2 °C).
  • Near-critical CO 2 can be used for extractions at temperatures that do not result in thermal degradation of sensitive components.
  • the use of near-critical CO 2 as a solvent for the extraction of totarol gives a high yield of totarol-containing extract, and leaves no solvent residues in the extract or spent wood, as compared with conventional extraction methods using organic solvents, in which considerable effort is required to reduce solvent residues to levels required to satisfy regulatory requirements.
  • the plant material is contacted with the near-critical fluid at a temperature in the range 273-373 K and at a pressure which is sufficient to ensure adequate recovery of the totarol-containing extract from the plant material.
  • the plant material is contacted with the near-critical fluid at a temperature in the range 273-373 K and at a pressure in the range 50-500 bar. More preferably, the temperature is in the range 273- 353 K and the pressure is in the range 50-300 bar. Still more preferably, the temperature is in the range 287-333 K and the pressure is in the range 70-300 bar.
  • the totarol-containing extract produced in a process of the present invention may also contain other bio-active and non-active totarol derivatives and associated compounds present in the plant material.
  • Other compounds which have been found to be present in the totarol-containing extract include: podototarin; totarol hemiacetal; 7a-hydroxytotarol; 7-oxototarol; 5-hydroxytotarol; 6,7-dehydrototarol; and ferruginol.
  • the extract may also contain other compounds including, but not limited to other known totarol derivatives such as: 19-hydroxytotarol; sugiol; methyl podocarpate; podocarpic acid; ⁇ -sitosterol; and pododacric acid (7).
  • totarol derivatives such as: 19-hydroxytotarol; sugiol; methyl podocarpate; podocarpic acid; ⁇ -sitosterol; and pododacric acid (7).
  • a degree of fractionation of the components present in the totarol-containing extract may be achieved by appropriate selection of the pressure and temperature at which the plant material is contacted with the near-critical fluid. Accordingly, in one embodiment, the plant material is contacted with the near-critical fluid at a first pressure to produce an extract having a higher proportion of totarol, followed by contacting at a second, higher, pressure to produce an extract with a relatively higher proportion of non-totarol components.
  • Suitable plant material includes the wood, bark, roots and leaves of those species known to contain totarol (7). These species include various Podocarpus, Dacrycarpus, Cupressus and Juniper'us species.
  • the plant material is obtained from Podocarpus totara G. Benn (Totara) or Podocarpus Hallii (Halls Totara), both of which are indigenous to New Zealand. More preferably, the plant material is heartwood obtained from these species.
  • totarol may be extracted from dead Totara wood including stumps, used fence posts and building timbers, and fallen tree logs recovered from the forest or swamps.
  • the plant material may be ground to a selected particle size, thereby increasing the surface area to volume ratio, prior to being contacted with a near-critical fluid in a process of the invention.
  • the particle size is between about 0.01 and 5 mm. More preferably, the particle size is between 0.01 and 3 mm. Still more preferably, the particle size is between 0.05 and 1 mm.
  • Water present in the plant material may be co-extracted into the near-critical fluid phase in which the totarol is dissolved. Accordingly, the totarol-containing extract produced after separation from the near-critical fluid may contain varying amounts of water. If required, this water may be removed by variety of means as would be appreciated by a person skilled in the art.
  • the totarol-containing extract is freeze-dried.
  • the freeze-dried extract may also be milled to a powder.
  • the plant material is partially to completely dried prior to being contacted with a near-critical fluid in a process of the invention.
  • Partially dry plant material will typically have a moisture content of ⁇ 15 % on a weight basis, while completely dry material will have a moisture content of ⁇ 2 % on a weight basis.
  • the use of completely dry plant material may render subsequent drying of the totarol-containing extract unnecessary.
  • the plant material may be ground to a selected particle size before drying.
  • the increased surface to volume ratio after grinding will increase the rate at which the plant material dries.
  • the plant material may be dried before being ground to a selected particle size.
  • the plant material may be subject to a preliminary grinding to a first selected particle size, followed by drying and a final grinding to a second selected particle size.
  • the totarol-containing extract is separated from the near-critical fluid by transferring the near-critical fluid phase in which the totarol is dissolved into a suitable separation vessel wherein the pressure is reduced to the point where the totarol-containing extract is precipitated into the vessel.
  • the pressure reduction can be carried out in a single step. Alternatively, the pressure reduction may be carried out in multiple steps to give more than one fraction of the totarol- containing extract.
  • the totarol-containing extract is separated from the near-critical fluid in several sequential pressure reductions and/or temperature changes, thereby fractionating the extract into a plurality of separation vessels.
  • the first pressure reduction is in a separation vessel held at a first selected pressure which is less than the pressure at which the plant material is contacted with a near- critical fluid, but greater than the pressure at which any subsequent separation vessel is held. More preferably, where the near-critical fluid is CO 2 , the first selected pressure is in the range 55-120 bar. Still more preferably, the first selected pressure is in the range 55-100 bar.
  • the second pressure reduction is in a separation vessel held at a second selected pressure which is less than the first selected pressure. More preferably, the second selected pressure is in the range 1-70 bar. Still more preferably, the second selected pressure is in the range 40-60 bar.
  • the separation vessel is equipped with a basket in which the totarol- containing extract is collected.
  • a basket in which the totarol- containing extract is collected.
  • Such baskets are well-known to those skilled in the art.
  • the use of a collection basket permits convenient emptying of the separation vessel.
  • the collection basket may be readily removed from the depressurised separation vessel while the pressure of the extraction vessel is maintained, thereby permitting semi-continuous operation of the apparatus.
  • one or more auxiliary separation vessels may be used to recover the totarol-containing extract, again permitting semi-continuous operation of the apparatus.
  • the totarol-containing extract may be subjected to a re-extraction at different near-critical conditions to provide a second totarol-containing extract in which the proportions of totarol and other components differ from those in the original extract.
  • totarol-containing extract produced from the extraction of plant material at relatively high pressures may be re- extracted at lower pressures to yield an extract in which the proportion of totarol has increased.
  • a carrier liquid is used to separate the totarol-containing extract and the near-critical fluid.
  • the carrier liquid is injected into the flow of the near-critical fluid phase in which the totarol is dissolved upstream of the separation vessel/s.
  • the carrier liquid dissolves the solid totarol-containing extract after the pressure is reduced to give an infusion of totarol in the carrier liquid, and prevents the solid totarol-containing extract from, for example precipitating in the lines and valves and causing blockages.
  • the use of a carrier liquid also enables the continuous removal of the infusion from the separation vessel/s without depressurisation.
  • the infusion may be recycled by removal from the separation vessel/s and re-injection into the flow of the near-critical fluid phase. Generally, the infusion may be recycled until the carrier is saturated with the totarol-containing extract, thereby providing a more concentrated infusion.
  • Suitable carrier liquids include oils but are not limited thereto. It will be appreciated that the carrier liquid must have a sufficiently low melting point such that it does not solidify in the apparatus.
  • the carrier liquid is an oil with a melting point less than about 10°C.
  • the oil may be pharmaceutically, nutraceutically, cosmetically or cosmeceutically acceptable, such that the infusion of the totarol-containing extract in the oil is suitable for formulation with, and into, pharmaceuticals, nutraceuticals, cosmetics or cosmeceuticals.
  • oils include vegetable oils, seed oils and nut oils. More preferably, the oil is selected from: soya oil; linseed oil; evening primrose oil; rosehip oil; borage oil; blackcurrant oil; kiwifruit seed oil; avocado oil; olive oil; and mixtures thereof.
  • the present invention provides a process for producing a totarol-containing extract, which extract comprises totarol and an oil, from suitable plant material and suitable oil-containing material, which process comprises the steps of: (a) contacting the plant material and oil-containing material with a near-critical fluid to produce a near-critical fluid phase in which the totarol and oil is dissolved;
  • Suitable oil-containing materials include seeds and other matter from which oil can be extracted.
  • solid totarol-containing extract of the invention may also be mixed with a suitable oil to provide an infusion comprising totarol.
  • the present invention provides a totarol-containing extract when produced by a process of the invention.
  • a totarol-containing extract which is free of solvent residues may be produced.
  • Such totarol-containing extracts are, therefore, suitable for use in the formulation of pharmaceuticals, nutraceuticals, cosmetics, cosmeceuticals and other products.
  • the present invention provides a composition comprising a totarol-containing extract produced by a process of the invention.
  • the totarol-containing extracts of the invention have antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, the totarol-containing extract of the present invention is contemplated for use as an active ingredient, antimicrobial additive or preservative in a variety of products including, but not limited to: household and industrial cleansers and disinfectants; cosmetic formulations; cosmeceuticals; pharmaceuticals; nutraceuticals; skin and body care products, such as soap, body wash, moisturiser and facial scrub; and dental care products, such as toothpaste, mouth spray and mouthwash.
  • the invention provides a method for at least inhibiting the growth of bacteria sensitive to the totarol-containing extract of the invention, the method comprising contacting the sensitive bacteria with an inhibitory effective amount of the totarol-containing extract of the invention, or a composition comprising the totarol-containing extract of the invention.
  • the invention provides a method of prophylactic or therapeutic treatment of bacterial infection in an individual in need thereof, the method comprising administering to said individual the totarol-containing extract of the invention, or composition comprising the totarol-containing extract of the invention, in an amount effective to at least inhibit growth of the bacteria.
  • the invention also provides a use of a totarol-containing extract of the invention in the preparation of a therapeutic formulation for treating a Gram-negative bacterial infection in an individual in need thereof.
  • the term "individual” as used herein includes humans, horses, dogs, cats, pigs, sheep, cattle, goats but is not limited thereto. Preferably, the individual is a human.
  • the present invention provides a therapeutic fonnulation having activity against Gram-positive and Gram-negative bacteria, which formulation comprises a totarol- containing extract produced by a process of the invention.
  • the totarol-containing extract of the invention comprises active nonvolatile solids and, as such, is particularly suitable for use in topical formulations where it will remain on the skin, and disinfectant applications, in comparison to those using other known natural anti-microbials such as tea-tree oil which may evaporate, thereby reducing their efficacy.
  • therapeutic formulations in which the totarol-containing extract of the invention can be employed include orally administrable medicaments such as: capsules; tablets; lozenges; syrups; mouthwashes; gargles; toothpastes; chewing gums; chewable tablets; and mouth sprays, but are not limited thereto.
  • Contemplated topical formulations include: lotions; creams; gels; sticks; sprays; shaving creams; ointments; cleansing liquid washes and solid bars; shampoos; pastes; powders; mousses; wipes; patches; nail lacquers; wound dressings; adhesive bandages; hydrogels; films; and make-up such as concealers, foundations, mascaras, and lipsticks.
  • a “therapeutic formulation” is a formulation appropriate for administration of the totarol- containing extract of the invention, to an individual in need of same.
  • therapeutic formulations of the invention comprise the totarol-containing extract of the invention and an acceptable carrier, diluent and/or excipient.
  • an "acceptable carrier, diluent and/or excipient” means a vehicle for delivery of the totarol- containing extract of the invention, to the individual, in which the vehicle is compatible with the extract.
  • Acceptable carriers, diluents and excipients suitable for use in the administration of the totarol-containing extract of the invention are well known to those skilled in the art. Suitable carriers are generally inert and can be either solid or liquid.
  • the carrier is a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers suitable for topical administration include, but are not limited to: solutions; emulsions such as microemulsions and nanoemulsions; gels; solids; and liposomes. Such carriers can be readily formulated by those skilled in the art.
  • a variety of pharmaceutically acceptable carriers suitable for oral administration are also well known in the art (8).
  • compositions may also include excipients such as tableting aids; resins; fillers; binders; lubricants; solvents; glidants; disintegrants; preservatives; buffers; flavourings; colourings; sweeteners; and fragrances as appropriate.
  • excipients such as tableting aids; resins; fillers; binders; lubricants; solvents; glidants; disintegrants; preservatives; buffers; flavourings; colourings; sweeteners; and fragrances as appropriate.
  • compositions of the invention may further comprise one or more secondary antimicrobial agents compatible with the totarol-containing extract of the invention.
  • the proportion of the totarol-containing extract in the formulations and compositions will vary depending on the contemplated end use.
  • the formulations and compositions of the invention typically may comprise from about 0.005 to 20% by weight of the totarol- containing extract.
  • the composition preferably comprises between about 0.005 and 1% totarol-containing extract.
  • the near-critical extractions were carried out in apparatus schematically depicted in Figure 1.
  • CO 2 (or other near-critical fluid) was supplied to the apparatus by liquid supply cylinders CYL1 and CYL2.
  • the apparatus included a high pressure pump MP3 for circulating the near-critical fluid, and two extraction vessels EX1 and EX2, each of which had a volume of 10 litres at pilot scale and 150 litres at demonstration scale.
  • the apparatus further included two separation vessels SN1 and SV2 which enabled the recovery of the totarol-containing extract in two fractions. Pumps MP1 and MP2 were used to refill the extraction vessels.
  • the apparatus also included a CO 2 recovery vessel RN1 and associated compressor RC1, together with pressure control valve CN1, back pressure regulator BPR, flow meter FM1, and various valves (V), indicators (P or T) and heat exchangers (HX).
  • the apparatus When oil was used as a carrier liquid to remove the totarol-containing extract from the near-critical CO , the apparatus further included an oil reservoir OT1 and an oil pump OP1.
  • Totara wood was ground in a knife mill to a desired particle size — typically between about 0.01 and 5 mm — and then added to a preweighed extraction basket having porous plates at either end to enable the passage of the near-critical fluid but not solvent.
  • the basket was weighed to determine the mass of Totara wood before extraction.
  • the basket was placed in extraction vessel EX1, which was then sealed and filled with near-critical fluid up to the cylinder pressure via valve NDF1.
  • EX1 was then raised to the operating pressure — typically 300 bar for CO 2 — initially using pump MP2 up to 70 bar and then using pump MP1 or pump MP3. When the desired pressure had been reached, flow was started through the vessel(s) and near-critical fluid was continuously circulated around the apparatus in a loop.
  • the pressure was reduced to a selected pressure — typically between 120 and 55 bar for CO 2 — through valve CN1 to effect separation of the bulk of the totarol-containing extract from the near-critical fluid.
  • the main totarol-containing extract fraction was precipitated into separation vessel SV1.
  • the near-critical fluid and remaining totarol-containing extract then passed through pressure control valve BPR1 and heat exchanger HX3 where the pressure was reduced to the cylinder pressure — typically between 40 and 60 bar for CO 2 — and the temperature was raised — to approximately 313 K for CO 2 .
  • Water, a small amount of totarol-containing extract, and essential oil was precipitated into vessel SN2.
  • the near- critical fluid, now a gas, was then recycled back to the main pump (MP1 or MP3) via heat exchanger HX4, water-trap WT1 and condenser-subcooler HX5 or HX1, respectively.
  • the separation vessel SNl was used in continuous mode wherein the totarol-containing extract was removed through valve EN1, or in batch mode wherein a collection basket (a canister with an open top) was placed in the vessel to collect the extract.
  • a collection basket a canister with an open top
  • Detection was at 210 nm and the method was calibrated using totarol purified by RPLC and pure by 1H ⁇ MR and RPLC— UN (MeOH) 207 nm ( log ⁇ 4.39). The quantities of the minor components isolated were insufficient to determine response factors, so quantification of these compounds assumed the same response factor as measured for totarol. The proportion of each compound in the sub-sample was calculated as a percentage by weight.
  • the testing protocol was as described above, except that the totarol extracts and tea-tree oil were combined at concentrations ranging from 0 to 10 ⁇ g or ⁇ L (respectively)/mL, and a kinetic assay was performed overnight in a micro-plate reader, set at 30°C. Each combination was analysed in duplicate.
  • Totara wood chip obtained from fence posts was subjected to near-critical extraction at a pilot scale according to the general procedure described above. Extractions were carried out without carrier liquid, at a variety of particle sizes, extraction pressures and temperatures. A two-stage separation was used, except where the near-critical fluid was liquid CO 2 at 70 bar and 287 K. The extraction conditions and yields of the totarol-containing extract are shown in Table 2. Table 2: Extract yield as a function of particle size and extraction conditions
  • Totara wood chips obtained from fence posts with a chip size between about 5 and 10 mm were partly to fully dried in metal trays in a convection oven at 80°C. The weight loss of the tray as a function of time was used to determine the percentage of moisture removed. Partly dried (moisture content of 5.8 %) and fully dried Totara wood chips (moisture content of 1.0 %) were then extracted in two separate trials at a pilot scale. The Totara wood chips comprised approximately 10 % volatile matter before drying, most of which was water. The wood chips were further ground in a knife mill to a particle size between about 0.5 and 1 mm after drying. The results of the extractions — both of which were carried out at 310 K — are summarised in Table 3.
  • Chip 1 st separation vessel 2 ,nd separation vessel Moisture CO 2 mass mass "extt”sep (%) (bar) (g) (kg) % yield % totarol % yield % totarol 3153.3 5.8 47.54 300/85 3.44 46 1.76 72 2802.0 1.0 47.25 300/85 3.46 40 2.07 68
  • This example shows how a carrier liquid — typically an oil — can be injected into the CO 2 stream flowing in the apparatus, prior to one of the separation vessels.
  • the oil dissolves the totarol-containing extract after it exits the depressurisation valve thereby preventing blockages.
  • the use of oil as a carrier liquid also enables the continuous removal of the extract from the separation vessels, and yields an oil infusion comprising the totarol- containing extract.
  • NM not measured
  • Oil mass oil injected into the flowing CO 2 just prior to the specified separation vessel
  • P ext extraction pressure
  • P sep separation pressure
  • the totarol-containing extracts were able to be continuously removed from the separation vessel downstream of the point of injection of the oil into the CO .
  • the infusion — comprising the oil and the totarol-containing extract— obtained from the second separation vessel was yellow to brown in colour, and contained water which separated out upon standing.
  • the totarol-containing extract was fully dissolved in the oil.
  • the oil infusion could be recycled, and re-injected prior to the separation vessel, until a totarol concentration of approximately 10 % by mass was reached.
  • the main extract from the demonstration scale extraction was freeze-dried in a tray freeze- drier.
  • totarol-containing extract produced at relatively high pressures can be re-extracted at different near-critical extraction conditions to yield an extract with a higher proportion of totarol.
  • This example shows that Totara wood chips obtained from fence posts can be extracted at a relatively low extraction pressure to yield a totarol-containing extract having a higher proportion of totarol than the extract produced at 300 bar, and that all of the totarol can be extracted from the wood chips.
  • This example further demonstrates the use of collection baskets in the separation vessels, and their exchange without depressurising the extractor. This example also shows that, following an extraction at relatively low pressure, the pressure can be raised to extract non-totarol compounds from the wood chips.
  • This example demonstrates the semi-continuous near-critical extraction of Totara wood chips.
  • the demonstration scale extraction of Totara wood chips — with a particle size between about 1 and 2 mm — obtained from fence posts was carried out using supercritical CO 2 at 150 bar and 60°C (333 K) without the use of a carrier liquid.
  • 335.4 kg of wood chips were processed with 8347 kg of CO 2 to give 14.35 kg of a mixture of totarol-containing extract and water in the first separation vessel, and 3.18 kg of a mixture comprising mainly water and a small amount of totarol-containing extract in the second separation vessel.
  • the extraction was carried out using three extraction vessels, one of which was being emptied and refilled at any time, and the other two of which were in the CO 2 flow.
  • Each vessel was on-line for four hours, and off-line — being emptied and refilled— for two hours.
  • Each of the extraction vessels held between approximately 40 and 50 kg of Totara wood chips.
  • the first separation vessel was equipped with a basket to collect the extract.
  • the separation vessel was operated for six hours before it was depressurised, the basket removed and replaced, and then repressurised. Free water was removed from the main product by filtration prior to weighing the extract.
  • the main product was then freeze-dried in a tray drier to give 10.05 kg of totarol-containing extract.
  • the composition of this extract is shown in Table 6.
  • This example shows that a totarol-containing extract having a relatively high proportion of totarol can be produced from the logs of fallen trees recovered from the forest floor or swamp.
  • a kiln-dried swamp log was coarsely ground in a wood chipper. The large chips were then ground in a knife mill to a particle size between about 1 and 2 mm. 3107.7 g of the ground chips were then extracted using supercritical CO 2 at a pressure of 150 bar and temperature of 333 K for a total of three hours.
  • a collection basket was placed in the first separation vessel, which was operated at 70 bar and 333 K, and the second separation vessel was operated at 55 bar and 313 K.
  • the extraction was carried out according to the method of Example 5, with some modifications.
  • the first extraction was carried out for 60 minutes after which the first separation vessel was depressurised, the collection basket removed and replaced, and the separation vessel repressurised.
  • the extraction vessel was maintained at full pressure with no flow during the exchange of collection baskets in the separation vessel.
  • Extraction was then carried out for a further 60 minutes at the same conditions, after which the collection basket in the separation vessel was again removed and replaced.
  • the extraction was then carried out for a further 60 minutes, and the collection basket in the first separation vessel was again removed and replaced.
  • Table 7 Extract yield and proportion of totarol from the extraction of Totara fallen tree log heartwood
  • This example shows how an auxiliary separation vessel can be used to recover the totarol- containing extract from the first separation vessel, thereby enabling semi-continuous extraction without the use of oil as a carrier liquid, or the use of collection baskets.
  • valve ENl was opened to induce flow of extract into the auxiliary separation vessel.
  • valves ENl and EN2 were closed, and the gas in the auxiliary separation vessel was then slowly vented to atmosphere.
  • the auxiliary separation vessel was then removed and replaced with a second auxiliary separation vessel.
  • the totarol-containing extract was recovered from the first auxiliary separation vessel by solvent extraction with acetone, followed by evaporation of the solvent under vacuum.
  • the second auxiliary separation vessel was then connected to the apparatus, and filled to the second separation vessel pressure of approximately 55 bar.
  • the auxiliary separation vessel was filled with extract in the same manner as the first auxiliary separation vessel after 120 minutes of extraction, and then the extraction was completed.
  • the second auxiliary separation vessel was removed as above and washed with acetone to recover the totarol- containing extract.
  • the first separation vessel was opened at the end of the extraction and was found to be empty of totarol-containing extract, confirming that all of the extract had passed into the auxiliary separation vessels.
  • MIC Minimum inhibitory concentration
  • the totarol-containing extract produced as the main extract in the demonstration scale extraction described in Example 3 was tested for activity against a range of Gram-positive bacteria using the agar dilution method.
  • the test samples were dissolved in ethanol, and then further diluted in water to give final concentrations, in a doubling dilution series, from 0.004 to 64 ⁇ g/mL.
  • the agar used was Mueller Hinton agar. An inoculum of approximately 1 x 10 4 organisms of each bacterial strain was replicated onto the agar plates containing the totarol-containing extract. The MIC for each bacterial strain is shown in Table 8.
  • Table 8 MIC of totarol-containing extract against Gram-positive bacteria
  • MIC Minimum inhibitory concentration
  • This example shows that the totarol-containing extract produced by supercritical extraction has antimicrobial activity similar to, or better than, tea-tree oil.
  • the MIC against four Gram-negative bacteria Salmonella menston; Escherichia coli; Enterobacter aerogenes; and Pseudomonas aeruginosa — was determined for the totarol- containing extract produced in Example 6, tea-tree oil and a mixture of the totarol- containing extract and tea-tree oil. The results are shown in Table 9.
  • Table 9 Comparison of the minimum inhibitory concentration (MIC) for totarol- containing extract, tea-tree oil, and a mixture of the extract and tea-tree oil against four Gram-negative bacteria
  • the results in Table 10 show that the totarol-containing extract is a very effective antimicrobial against a range of Gram-negative bacteria.
  • the MIC for each bacterial strain tested is approximately 0.3-1.25 ⁇ g/mL, and surprisingly, this figure is the same for each strain, whether subjected to the totarol-containing extract or tea-tree oil.
  • Tea-tree oil is known to have activity against Gram-negative bacteria, whilst pure totarol has not been reported to have any activity against Gram-negative bacteria.
  • Other components of the totarol-containing extract may be responsible for the activity against Gram-negative bacteria.
  • the antimicrobial effect of the totarol-containing extract is enhanced when combined with tea-tree oil only for Enterobacter aerogens.
  • a concentration of 0.3-0.6 ⁇ g/mL of the totarol-containing extract was sufficient to stop the growth of the other three Gram-negative strains tested. This concentration was not decreased by the addition of tea-tree oil, nor was the MIC of tea-tree oil affected by the addition of totarol-containing extract.
  • the results obtained for tea-tree oil are in accordance with those found in the literature, where the MIC for tea-tree oil ranges between 0.2 and 2.0 % (v/v), which equates to between 2.0 and 20 ⁇ L/mL.
  • the totarol-containing extract produced according to the methods described above was used to prepare a solution, suitable for topical application, and having the following composition:
  • a stock solution of FDC Blue No. 1 was prepared by weighing 10.5 mg into a 50 mL volumetric flask. The dye was dissolved in a small quantity of purified water and the solution made up to the mark with purified water.
  • the totarol-containing extract was dissolved in the ethyl alcohol and the propylene glycol added to the resulting solution.
  • a portion of the FDC Blue No. 1 solution (2.54 mL) was then added to give a solution with a green colour.
  • the purified water was then added in a sufficient quantity to give a total volume of 1 L. The resulting solution was transferred into applicator bottles.

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Abstract

L'invention concerne un procédé pour produire un extrait contenant du totarol à partir d'un végétal approprié. Ce procédé utilise un fluide quasi critique comme solvant d'extraction. Ce procédé est particulièrement applicable à l'extraction de totarol à partir de bois de totara. Cet extrait contenant du totarol possède une activité contre les bactéries à Gram positif et à Gram négatif. Cet extrait est produit sans résidus de solvant et convient, ainsi, à une utilisation dans des formulations pharmaceutiques, nutraceutiques, cosmétiques, cosméceutiques et autres.
PCT/NZ2005/000008 2004-01-28 2005-01-28 Procede d'extraction de totarol et/ou produit contenant du totarol WO2005073154A1 (fr)

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EP2143703A1 (fr) 2008-07-08 2010-01-13 Cognis IP Management GmbH Procédé de préparation de (+)-totarol
US20100056615A1 (en) * 2006-11-24 2010-03-04 Daniel Raederstorff Use of tricyclic diterpenes and their derivatives for the treatment, co-treatment or prevention of inflammatory disorders and/or joint disorders
CN101161324B (zh) * 2006-10-11 2012-07-04 韩延欣 亚临界流体萃取溶剂的萃取方法
WO2013089721A1 (fr) * 2011-12-15 2013-06-20 Colgate-Palmolive Company Composition antibactérienne à base de thymol et de totarol
WO2014185792A1 (fr) * 2013-05-17 2014-11-20 Ozospa Holdings Limited Produit de soins bucco-dentaires
DE102013108870A1 (de) 2013-08-16 2015-02-19 Aimecs Gmbh Mittel zur Reinigung und zum Schutz von technischen Oberflächen
WO2019009739A1 (fr) 2017-07-06 2019-01-10 Emergopharm Sp. Z O.O. Sp.K. Application de totarol et composition pharmaceutique contenant du totarol
CN114181051A (zh) * 2021-12-14 2022-03-15 广州杨森药业有限公司 一种桃柁酚的制备方法及其应用

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101161324B (zh) * 2006-10-11 2012-07-04 韩延欣 亚临界流体萃取溶剂的萃取方法
US20100056615A1 (en) * 2006-11-24 2010-03-04 Daniel Raederstorff Use of tricyclic diterpenes and their derivatives for the treatment, co-treatment or prevention of inflammatory disorders and/or joint disorders
EP2143703A1 (fr) 2008-07-08 2010-01-13 Cognis IP Management GmbH Procédé de préparation de (+)-totarol
RU2563208C1 (ru) * 2011-12-15 2015-09-20 Колгейт-Палмолив Компани Антибактериальная композиция, содержащая тимол и тотарол
WO2013089721A1 (fr) * 2011-12-15 2013-06-20 Colgate-Palmolive Company Composition antibactérienne à base de thymol et de totarol
AU2011383313A1 (en) * 2011-12-15 2014-06-19 Colgate-Palmolive Company Thymol and totarol antibacterial composition
AU2011383313B2 (en) * 2011-12-15 2014-07-31 Colgate-Palmolive Company Thymol and totarol antibacterial composition
US9832994B2 (en) 2011-12-15 2017-12-05 Colgate-Palmolive Company Thymol and totarol antibacterial composition
WO2014185792A1 (fr) * 2013-05-17 2014-11-20 Ozospa Holdings Limited Produit de soins bucco-dentaires
US20160120793A1 (en) * 2013-05-17 2016-05-05 Alequident Limited Oral Healthcare Product
DE102013108870A1 (de) 2013-08-16 2015-02-19 Aimecs Gmbh Mittel zur Reinigung und zum Schutz von technischen Oberflächen
WO2019009739A1 (fr) 2017-07-06 2019-01-10 Emergopharm Sp. Z O.O. Sp.K. Application de totarol et composition pharmaceutique contenant du totarol
CN114181051A (zh) * 2021-12-14 2022-03-15 广州杨森药业有限公司 一种桃柁酚的制备方法及其应用
CN114181051B (zh) * 2021-12-14 2024-05-07 广州杨森药业有限公司 一种桃柁酚的制备方法及其应用

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